110 12 Generation and differentiation of human iPS cells Sebastian Diecke, 1 Lei Ye, 2 Sophia Zhang, 2 and Jianyi Zhang 2 1 Stanford University School of Medicine, Palo Alto, CA, USA 2 University of Minnesota Medical School, Minneapolis, MN, USA Introduction Perhaps one of the most signiicant achievements in science was the discovery of induced pluripotent stem (iPS) cells [1,2]. iPS cells are a type of pluripotent stem cell derived from adult somatic cells that have been genetically reprogrammed back to an embryonic stem-cell-like state through the forced expression of genes and factors important for maintaining the deining properties of embry- onic stem (ES) cells [3]. Human iPS cells were irst produced from human ibroblasts by two independent groups – the Yamanaka lab at Kyoto University and the Thomson lab at University of Wisconsin, Madison. iPS cells are similar to ES cells in many aspects, including the expression of ES cell markers, chromatin methylation patterns, embryoid body formation, teratoma formation, viable chimera formation, and pluripotency in differentiation. The basic concept of somatic cell reprogramming was discovered in the late nineties through the cell fusion and somatic cell nuclear transfer experiments [4–6]. Based on these indings, Shinya Yamanaka and his coworkers were able to show that mouse embryonic ibroblasts (MEFs) could be converted into germline-competent induced pluripotent stem cells by retroviral expression of four transcription factors: OCT4, SOX2, KLF4, and c-MYC [3,7]. The reprogramming process is based on the ectopic expression of these transcription factors, which initiate a rare reactivation of the endogenous pluripotency genes OCT4, SOX2, and NANOG [8]. This reactivation leads to a resetting of the epigenetic proile of a terminally differentiated somatic cell and activates the molecular circuitry of pluripotency [9,10]. Thereafter, the derived iPS cells are similar to embryonic stem cells with regard to their morphology, growth characteristics, differentiation capacities, and expression of various marker genes [11]. The transition from a somatic to a pluripotent state could be further enhanced through chromatin- modifying chemicals and microRNAs [12,13]. Subsequent studies revealed that the induction of pluripotency was also possible with different transcription factor combinations and could be improved by activation or inhibition of different signaling pathways [14–18]. The unique property of iPS cells to differentiate into all other cell types of the human body opened up new clinical perspectives for developing successful stem-cell-based therapies. However, the use of retroviral and lentiviral vectors for reprogramming carries a high risk of DNA incorporation into chromosomes and can lead to the disruption of gene transcription and tumor formation [19]. Therefore, it is important to develop further nonintegrating reprogramming techniques for future applications in the ield of regenerative Manual of Research Techniques in Cardiovascular Medicine, First Edition. Edited by Hossein Ardehali, Roberto Bolli, and Douglas W. Losordo. © 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd.